Coherent Raman scattering microscopy (abbreviated “CRSM”) has recently acquired considerable significance in image-producing chemical sample analysis, for example in biology, pharmacy, or food science. A variety of CRSM methods are utilized, for example coherent anti-Stokes Raman scattering (CARS), coherent Stokes-Raman scattering (CSRS), Raman-induced Kerr effect scattering (RIKES), and stimulated Raman scattering (SRS). The list of documents below will be referred to hereinafter regarding the existing art:    [1] Nandakumar, P., Kovalev, A., Volkmer, A.: “Vibrational imaging based on stimulated Raman scattering microscopy,” New Journal of Physics, 2009, 11, 033026.    [2] Freudiger, C. W., Roeffaers, M. B. J., Zhang, X., Saar, B. G., Min, W., Xie, X. S.: “Optical heterodyne-detected Raman-induced Kerr effect (OHD-RIKE) microscopy,” Journal of Physical Chemistry B, 2011, 115, 5574-5581.    [3] Saar, B. G., Freudiger, C. W., Reichman, J., Stanley, C. M., Holtom, G. R., Vie, X. S.: “Video-rate molecular imaging in vivo with stimulated Raman scattering,” Science, 2010, 330, 1368-1370.    [4] Mikhail N. Slipchenko, Robert A. Oglesbee, Delong Zhang, Wei Wu, Ji-Xin Cheng: “Heterodyne detected nonlinear optical imaging in a lock-in free manner,” J. Biophotonics, 2012, 5, 1-7.    [5] Zumbusch, A., Holtom, G. R., Xie, X. S.: “Three-dimensional vibrational imaging by coherent anti-Stokes Raman scattering,” Phys. Rev. Lett., 1999, 82, 4142-4145.    [6] Cheng, J. X. and Xie, X. S.: “Coherent anti-Stokes Raman scattering microscopy: Instrumentation, theory, and applications,” J. Phys. Chem. B, 2004, 108, 827-840.    [7] Evans, C. L. and Xie, X. S.: “Coherent anti-Stokes Raman scattering microscopy: chemical imaging for biology and medicine,” Annu. Rev. Anal. Chem., 2008, 1, 883-909.    [8] Dudovich, N., Oron, D., Silberberg, Y.: “Single-pulse coherently controlled nonlinear Raman spectroscopy and microscopy,” Nature, 2002, 418, 512-514.    [9] Hellerer, T., Enejder, A. M. K., Zumbusch, A.: “Spectral focusing: High spectral resolution spectroscopy with broad-bandwidth laser pulses,” Applied Physics Letters, 2004, 85, 25-27.    [10] Israel Rocha-Mendoza, Wolfgang Langbein, Paola Borri: “Coherent anti-Stokes Raman microspectroscopy using spectral focusing with glass dispersion,” Applied Physics Letters, 2008, 93, 201103:1-201103:3.    [11] Adrian F. Pegoraro, Andrew Ridsdale, Douglas J. Moffatt, Yiwei Jia, John Paul Pezacki, Albert Stolow: “Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator,” Optics Express, 2009, 17, 2984-2996.
In the CRSM technique, two pulsed light fields having pulse widths in a range from 100 fs to 20 ps, of different wavelengths, are directed through a confocal microscope optical system and focused onto the sample. The pulsed light fields, having frequencies that are typically in a range from 1 to 100 MHz, are emitted from a short-pulse laser light source. The light fields are spatially and temporally superimposed on one another on the sample via corresponding beam guidance and suitable focusing optics. “Temporal superimposition” is to be understood as pair-wise coincidence of the laser pulses forming the pulsed light fields. In the SRS method or the image-producing superimposed RIKES method, for example, one of the two light fields is modulated in terms of intensity, frequency, or polarization at a specific frequency that is typically in the kHz to MHz range, before interacting in the sample with the other light field. For SRS and RIKES image production, the initially unmodulated light field is then sensed and, using a lock-in technique or envelope curve demodulation technique, the intensity modulation is extracted and presented in the form of an image. Reference is made to documents [1], [2], and [3] regarding implementation of the lock-in technique. The envelope curve demodulation technique is described in document [4]. In the case of CARS and CSRS a third light field is sensed as a result of interaction with the sample and displayed as an image. This is described in documents [5], [6], and [7].
In all the CRSM techniques recited above, the measured signal is only strong if the difference between the frequencies of the incident light fields coincides with a vibrational resonance frequency in the sample. At present, the best images in terms of spectral selectivity, signal intensity, and signal to noise ratio are obtained using picosecond laser light sources and optical parametric oscillators (OPOs) having pulse widths from 5 to 7 ps.
Femtosecond lasers are also widely used nowadays, however, for example in multi-photon fluorescence microscopy and in microscopy techniques based on the principle of frequency doubling (SHG) or frequency tripling (THG). Considerable effort is therefore being devoted to using femtosecond lasers in CRSM imaging as well. Reference is made in this regard, for example, to documents [8], [9], [10], and [11].
The use of a femtosecond laser or of an optical parametric oscillator for CRSM has the considerable disadvantage, however, of poor spectral selectivity. This will be explained below with reference to FIGS. 1 and 2, in which the CARS emission achieved by excitation with a picosecond laser (FIG. 1) is compared, by way of example, with the CARS emission achieved by excitation with a femtosecond laser.
FIG. 1 shows, purely schematically, a term diagram of a sample that has a vibrational ground state |g> and a state system |v|> having a set of first excited vibrational states a, b, c. These first excited vibrational states a, b, c have energy levels that are attributable to three different molecular bonds, e.g. N—H, O—H, and C—H. In the CARS method two laser beams, one of which is referred to as a “pump beam” and the other as a “Stokes beam,” are directed onto the sample. The energy difference between these beams coincides with the energy of one of the vibrational states. A probe beam, which probes the vibrational coherence, is also used.
In the case shown in FIG. 1, in which CARS emission is excited with a picosecond laser, the pump beam and the Stokes beam are spectrally comparatively narrow-band. This is due to the circumstance, known from Fourier analysis, that as the extent of a laser pulse over time increases, the spectral distribution of the frequencies contained in the laser pulse becomes narrower. It is accordingly possible to selectively excite the vibrational states a, b, c using the picosecond laser.
FIG. 2, in contrast, illustrates the corresponding situation when a femtosecond laser is used instead of the picosecond laser. Because the time-related pulse widths of the pump beam and Stokes beam are smaller in this case, the spectral widths of the laser pulses correspondingly increase. It is consequently no longer possible to selectively excite the individual vibrational states a, b, c. Spectral selectivity is thus negatively affected by the spectrally broad-band excitation of the femtosecond laser.
A variety of methods, known in the literature under the keywords “spectral focusing,” have been proposed as a solution to this problem. Reference is made in this regard to document [10]. In this, two glass blocks of predetermined length are used, one of which is arranged in the light path of the Stokes beam while the other is located in a shared light path into which the Stokes beam and pump beam are combined. Each of these two glass blocks brings about, as a result of dispersion, a spectral broadening of the laser pulse passing through it. The glass blocks thus form so-called “chirp” units. The term “chirp” is to be understood here as a frequency-modifying influencing of the laser light, which can bring about e.g. a time-related stretching of the respective laser pulse but also a time-related compression of the laser pulse.
In the arrangement known from document [10], the dispersive effects of the two glass blocks as a function of the wavelengths of the pump beam and of the Stokes beam are selected so that the pertinent laser pulses, superimposed on one another on the sample, are coordinated with one another in the desired fashion, spectrally and in terms of time, in order to achieve the desired spectral focusing. For this, the glass blocks used as chirp units must be configured exactly for the wavelengths of the pump beam and Stokes beam. If other wavelengths are to be used, the glass blocks must be replaced with correspondingly modified units. This is costly and involves considerable complexity for the user.